The present invention relates to the synthesis and placement of materials at known locations. In particular, one embodiment of the invention provides a method and associated apparatus for the selective application of an array of oligonucleotides on a substrate by way of standard dimethoxytrityl (DMT) based chemistry. The invention may be applied in the field of preparation of an oligomer, a peptide, a nucleic acid, an oligosaccharide, a phospholipid, a polymer, or a drug congener preparation, especially to create sources of chemical diversity for use in screening for biological activity.
Industry utilizes or has proposed various techniques to synthesize arrays of oligonucleotides. One such technique is the use of small rubber tubes as reaction chambers to make up a single dimensional array by the sequential addition of reagents. This technique has advantages by the use of standard DMT based chemistry. However, a limitation with resolution often exists with such technique. Typically the smallest cell size is about 1 millimeter in dimension. This method also does not enable the synthesis of a sufficiently large number of polymer sequences for effective economical screening. A further limitation is an inability to form an array of, for example, oligonucleotides at selected regions of a substrate.
Other representative techniques are described in U.S. Pat. No. 5,143,854 and WO93/09668 which is hereby incorporated by reference for all purposes. Such techniques are finding wide use and are considered pioneering in the industry. In some applications, however, it is desirable to have alternative techniques and chemistries for synthesis of compound libraries.
It would be desirable to have a method and apparatus for making high density arrays of oligonucleotides using DMT-based chemistry and other suitable oligonucleotide synthesis chemistries, as is a method and apparatus for conventional phosphoramidite-based synthesis of a spatially defined array of oligomers (e.g., polynucleotides, polypeptides, oligosaccharides, and the like) each having a substantially predetermined sequence of residues (i.e., polymerized monomer units).
According to the present invention, a method and apparatus to form an array of polymers, such as oligonucleotides and a substrate using conventional linkage chemistries (e.g., standard DMT-based oligonucleotide synthesis chemistry) is provided. The method and apparatus includes use of selected printing techniques in distributing materials such as barrier materials, deprotection agents, base groups, nucleosides, nucleotides, nucleotide analogs, amino acids, imino acids, carrier materials, and the like to selected regions of a substrate. Each of the printing techniques may be used in some embodiments with, for example, standard DMT-based chemistry for synthesis of oligonucleotides, and in particular selected deprotecting agents in vapor form.
In a specific embodiment, the present invention provides a method of forming polymers having diverse monomer sequences on a substrate. In an embodiment, the method is used to synthesize oligonucleotides having predetermined polynucleotide sequence(s) on a solid substrate, typically in the form of a spatially defined array, wherein the sequence(s) of an oligonucleotide is positionally determined. The present method includes steps of providing a substrate with a linker molecule layer thereon. The linker molecule layer has a linker molecule and a protective group. The present method also includes a step of applying a barrier layer overlying at least a portion of the linker molecule layer. The barrier layer shields the underlying portion from contact with a reagent capable of otherwise reacting with the underlying portion and applied subsequent to application of the barrier layer, thereby substantially precluding a predetermined chemical reaction from occurring on areas of the substrate overlaid with the barrier material. The applying step forms selected exposed regions of the linker molecule layer. A step of exposing the selected exposed regions of the linker molecule layer (e.g., regions not overlaid with the barrier material) to a reagent, typically in vapor phase, and often comprising a deprotecting agent is also included.
In an alternative specific embodiment, the present method includes a method of applying a medium in selected regions of a substrate. The present method includes steps of providing a substrate with a top surface, and selectively applying a medium having an element selected from a group consisting of a barrier material, a receptor, a deprotection agent, a monomer group, a carrier material, and an activator to selected regions of the substrate top surface.
In an embodiment, the invention provides a method for synthesizing a spatial array of polymers of diverse monomeric sequence (e.g., such as a collection of oligonucleotides having unique sequences), wherein the composition (e.g., nucleotide sequence) of each polymer is positionally defined by its location in the spatial array. In general, the method employs a masking step whereby a spatially distributed barrier material is applied to a substrate to block at least one step of a monomer addition cycle from occurring on a portion of the substrate overlaid by the barrier material. The method comprises applying a barrier material to a first spatially defined portion of a substrate, said substrate optionally also comprising a layer of linker molecules and/or nascent polymers (e.g., nascent oligonucleotides), whereby the barrier material overlaying said first spatially defined portion of said substrate shields the underlying portion from contact with a subsequently applied reagent capable of otherwise reacting with the underlying portion and necessary for a complete monomer addition cycle whereby a monomer unit is covalently linked to a nascent polymer or linker, thereby substantially precluding a chemical reaction from occurring on said first spatially defined portion which is overlaid with the barrier material and providing a remaining unshielded portion of said substrate (i.e., portion(s) not overlaid with the barrier material) available for contacting said subsequently applied reagent and undergoing said chemical reaction necessary for a complete monomer addition cycle (i.e., polymer elongation). The subsequently applied reagent is typically a monomer (e.g., nucleotide, nucleoside, nucleoside derivative, amino acid, and the like), a deprotecting agent for removing protecting group(s) which block polymer elongation (e.g., removal of DMT groups by acid hydrolysis), a coupling agent (e.g., phosphoramidites, such as cyanoethyl phosphoramidite nucleosides), a capping agent (e.g., acetic anhydride and 1-methylimidazole), and/or an oxidation agent (e.g., iodine; such as in iodine:water:pyridine:tetrahydrofuran mixture). The method further provides that, subsequent to the application of the barrier material, the reagent(s) is/are applied and permitted to chemically react with the unshielded portion of the substrate for a suitable time period and under suitable reaction conditions. Following reaction of the unshielded portion with the reagent(s), monomer addition is completed and the barrier material is removed (not necessarily in that order), resulting in a monomer addition to polymer(s) in the unshielded portion of the substrate and substantial lack of monomer addition to polymer(s) in the shielded portion of the substrate, during said monomer addition cycle.
In an embodiment, the masking step, wherein a barrier material is applied to a spatially defined portion of the substrate and used to shield said spatially defined portion to block a monomer addition cycle on said spatially defined portion, is employed repetitively. A first barrier mask is applied to overlay a first spatially defined portion of a substrate creating: (1) a first shielded portion overlain by said barrier mask, and (2) a first unshielded portion comprising the portion of the substrate not overlain by said barrier mask. The application of the first barrier mask is followed by completion of a first monomer addition cycle, whereby a monomer unit is covalently added to the first unshielded portion to extend or initiate a nascent polymer bound to said substrate, typically covalently, and whereby said first monomer addition cycle substantially fails to result in addition of a monomer unit to nascent polymers in the first shielded portion. The first barrier mask is removed, concomitant with, prior to, or subsequent to the completion of said first monomer addition cycle, and one or more subsequent cycles of applying a subsequent barrier mask, which may overlay subsequent shielded portions which is/are spatially distinct from said first shielded portion, and performing at least one subsequent monomer addition cycle(s) followed after each cycle by barrier removal, and optionally, reapplication of a barrier mask and initiation of a further monomer addition cycle until polymers of a predetermined length (number of incorporated monomer units) are produced.
In an aspect of the invention, a repetitive masking/synthesis process can be comprised of the following steps:
(1) application of barrier material to substrate having a reactive surface capable of covalently bonding to a monomer unit or reacting with a deprotecting agent or other reagent necessary for completion of a monomer addition cycle, said reactive surface being derivatived with a linker and/or a monomer unit or nascent polymer (e.g., a 3xe2x80x2-linked nucleoside or 3xe2x80x2-linked polynucleotide), wherein said barrier material covers a portion of said reactive surface creating a covered portion, said covered portion being a shielded portion and being substantially incapable of reacting with a monomer unit or reagent necessary for completion of a monomer addition cycle, and the remaining portion of the substrate being an unshielded portion capable of reacting with a monomer unit or reagent necessary for completion of a monomer addition cycle;
(2) contacting the substrate with reagents necessary for completion of a monomer addition cycle, wherein a monomer unit is covalently attached to the reactive surface of the substrate (e.g., a linker, a 3xe2x80x2-linked nucleoside, or 3xe2x80x2-linked nascent polynucleotide) in an unshielded portion;
(3) removing the barrier material; and
(4) repeating steps 1, 2, and 3 from 0 to 5000 cycles, preferably from 2 to 250 cycle, more usually from 4 to 100 cycles, and typically from about 7 to 50 cycles, until a predetermined polymer length is produced on a portion of the substrate. The pattern of barrier material applied in each cycle may be different that the prior or subsequent cycle(s), if any, or may be the same. Often, in step (2), at least one reagent necessary for completion of a monomer addition cycle is applied in vapor phase.
In an embodiment of the invention is provided a substrate having a spatial array of polymers of predetermined length produced by the method described supra.
In one aspect of the invention is provided a method for applying a barrier material or reagent necessary for a monomer addition cycle to a substrate, said method comprising transferring the barrier material or reagent as a charged droplet by electrostatic interaction, such as, for example, in an inkjet or bubble jet print head or similar device. In an embodiment, the barrier material or reagent is suitable for use in polynucleotide (oligonucleotide) synthesis. In an embodiment, the substrate is a silicon or glass substrate or a charged membrane (e.g., nylon 66 or nitrocellulose).
An aspect of the invention provides a method for synthesizing polynucleotides on a substrate, said method comprising application of at least one reagent necessary for addition of a nucleotide to a nascent polynucleotide or linker molecule bound to a substrate, wherein said application is performed with the reagent present substantially in vapor phase.
A further understanding of the nature and advantages of the present invention may be realized by reference to the latter portions of the specification and attached drawings.